1. Field of the Invention
The present invention relates to a method of reducing the content of tobacco specific nitrosamines (hereinafter referred to as “TSNA”) in tobacco leaves. More particularly, the invention relates to a method of reducing TSNA content in the tobacco leaves by inhibiting microbial growth involved in production of nitrite, a precursor of TSNA.
2. Description of the Related Art
TSNA contained specifically in cured tobacco leaves are not present in tobacco leaves immediately after harvest; however, during the curing process and storage process thereafter, TSNA are formed by reaction of nitrite and alkaloids contained in the tobacco leaves. The main components of TSNA formed in such a manner are N-nitrosonornicotine (hereinafter, referred to as “NNN”), 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (hereinafter, referred to as “NNK”), N-nitrosoanatabine (hereinafter, referred to as “NAT”), N-nitrosoanabasine (hereinafter, referred to as “NAB”), and the like.
The varieties of tobacco cultivated in Japan are broadly classified into three groups; flue-cured tobacco, Burley tobacco, and Japanese domestic tobacco.
The harvested tobacco leaves are green, but chlorophyll in the plant cell is degraded and carotenoide pigment appears during curing process. The carotenoide pigment is a yellow color pigment and thus the color of the tobacco leaves turns to be yellow.
With respect to the flue-cured tobacco, after the tobacco leaves turn to be yellow, the speed of dehydration is quickened by raising the curing temperature, and finally the color of the cured leaves is fixed to be yellow.
On the other hand, with respect to Japanese domestic and Burley tobaccos, the curing process still continues after yellowing stage, and during the continuous curing stage, the carotenoide pigment is degraded and a brown pigment is produced to turn tobacco leaves to be brown. After that, the lamina and stem are completely dried and the curing process is finished. As described, the Burley and Japanese domestic tobacco leaves turn to be cured leaves through yellowing, browning, and stem drying stages.
The flue-cured tobacco and the Burley and Japanese domestic tobaccos differ in the curing methods. In the case of curing the flue-cured tobacco, harvested tobacco leaves are hung in a curing barn (a bulk curing barn) equipped with a heater, and cured while the temperature and humidity being controlled by using wind and fire powers, so that the tobacco leaves are cured in 5 to 7 days through the yellowing stage, color-fixing stage, and stem drying stage. On the other hand, in the case of curing the Burley and Japanese domestic tobaccos, harvested tobacco leaves are hung in a pipe house or a wooden curing house and cured while the temperature and humidity being controlled mainly in natural conditions, so that the tobacco leaves are cured in 25 to 35 days through the yellowing stage, browning stage, and stem drying stage.
Such curing of the tobacco leaves is carried out aiming not only to dry the tobacco leaves but also to convert the components in the tobacco leaves and provide colors, flavor and taste that are specific to the tobacco varieties. Thereafter, for maturing further flavor and taste, the tobacco leaves that have been finished the curing process are stored. However, during such curing and storage processes, the formation of TSNA is caused by a reaction of nitrite with alkaloids contained in the tobacco leaves. In the case of flue-cured tobacco, TSNA are formed mainly during curing by heating and in the case of Burley tobacco, TSNA are formed from the browning stage to stem drying stage in the curing processing steps.
It has been known that laminas of tobacco leaves immediately after harvest contain amino acids, proteins, and alkaloids as well as nitrate and nitrite. Generally, plants produce amino acids from nitrate via nitrite in vivo and utilize the amino acids for formation of the plant. On the other hand, since nitrite in a high concentration causes adverse effects on life of the plant, plants synthesize only in the minimum amounts required for utilization for the plant formation. Accordingly, the content of the nitrite-nitrogen in the tobacco leaves is 1 ppm or lower immediately after harvest.
However, during the curing process of the tobacco leaves, because of the function of nitrate reducing enzymes produced by microorganisms existing in the tobacco leaf surface, the nitrate in the tobacco leaves is reduced to nitrite. The produced nitrite is reacted with alkaloids in the tobacco leaves, so that TSNA are formed and accumulated in the leaves.
Conventionally, various techniques for reducing the TSNA content in the tobacco leaves have been proposed and for example, there have been proposed as follows.
In terms of cultivation of tobacco, there is a method of decreasing the amount of a nitrogen fertilizer to be used. Decrease of the amount of the nitrogen fertilizer reduces the alkaloid content in the leaves, which are origin substances of TSNA formation. It has been proved that the TSNA content in the leaves is decreased by the method.
In terms of plant breeding, new varieties having less alkaloid content in the leaves have been developed. In such development, seeds are taken out of plants having less alkaloid content and cultivated, so that varieties having a low TSNA content can be obtained.
With respect to flue-cured tobacco, there is proposed a method of reducing TSNA content by adopting an indirect-heating type of curing barn in place of a direct-heating type of curing barn. In this method, use of the indirect-heating type of curing barn reduces NOx, a precursor of TSNA, derived from fuel, so that the TSNA production is suppressed during the curing process (US Patent Application Publication No. US 2001/386).
Further, there is proposed a method of rapidly dehydrating and completing the curing process by treating tobacco leaves having a low TSNA content in the yellowing stage of the initial curing process with microwave (WO 98/05226). However, the method finishes curing in the middle of the conventional curing process and results in insufficient change in the components contained in the leaves. Thereby, the purpose of the curing is not accomplished, and it is impossible to exhibit characteristic color, flavor and taste. Accordingly, there occurs a problem that the flavor and taste of the tobacco leaves which have been cured more rapidly is deteriorated as compared with those of the tobacco leaves cured by a conventional method.
To inhibit reduction of nitrate in the tobacco leaves to nitrite by the function of the nitrate-reducing enzymes produced by microorganisms existing in the tobacco leaf surface during the curing process of the tobacco leaves, there is proposed a method of removing the relevant microorganisms in the tobacco leaf surface. For example, a method of washing out the microorganisms with bicarbonate of soda (WO 01/35770), a method of killing microorganisms with chlorine dioxide gas (WO 02/13636), and the like have been know.
Also, a denitrification treatment of the tobacco cured leaves by using a microorganism derived from tobacco leaves (WO 83/01180) is disclosed. However, the method makes it possible to decrease the content of nitrate and nitrogen compounds in the tobacco cured leaves but is insufficient to efficiently reduce TSNA content.
The inventors of the present invention have proposed a method of using TSNA-degrading bacteria as the method of reducing TSNA content in the tobacco leaves during the curing and storage processes (WO 03/094639).